Design and characterization of an APC-specific serpin for the treatment of hemophilia.

Hemophilia is a bleeding disorder caused by deficiency in factors VIII or IX, the two components of the intrinsic Xase complex. Treatment with replacement factor can lead to the development of inhibitory antibodies, requiring the use of bypassing agents such as factor VIIa and factor concentrates. An alternative approach to bypass the Xase complex is to inhibit endogenous anticoagulant activities. Activated protein C (APC) breaks down the complex that produces thrombin by proteolytically inactivating factor Va. Defects in this mechanism (e.g. factor V Leiden ) are associated with thrombosis, but result in less severe bleeding when coinherited with hemophilia. Selective inhibition of APC might therefore be effective for the treatment of hemophilia. The endogenous inhibitors of APC are members of the serpin family, protein C inhibitor (PCI) and α 1 -antitrypsin (α 1 AT), however, both exhibit poor reactivity and selectivity for APC. We mutated residues in and around the scissile P1-P19 bond in PCI and α 1 AT, resulting in serpins with the desired specificity profile. The lead candidate was shown to promote thrombin generation in vitro , and to restore fibrin and platelet deposition in an intravital laser injury model in hemophilia B mice. The power of targeting APC was further demonstrated by the complete normalization of bleeding after a severe tail clip injury in these mice. These results demonstrate that the protein C anticoagulant system can be successfully targeted by engineered serpins, and that administration of such agents is effective at restoring hemostasis in vivo .